DNA probe synthesis on chip on demand by MEMS ejector array

ABSTRACT

A DNA probe synthesis system may include a target holder for holding a target chip, an ejector array chip, a wash and dry station, and conveyance means for moving the target holder and the chip between the ejector array chip and the wash and dry station. The ejector array chip may include ejectors, reservoirs for containing DNA bases, and microchannels all on the same substrate. The ejectors may be Self Focusing Acoustic Transducers (SFATs).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/304,841, filed on Jul. 11, 2001.

BACKGROUND

A DNA microarray, or genome chip, may include hundreds of thousands ofDNA probes. DNA probes may include a known DNA sequence which may beused to recognize longer, unknown DNA sequences. The recognition ofsample DNA by the set of DNA probes on a glass wafer (or chip) takesplace through the mechanism of DNA hybridization. When a DNA samplehybridizes with an array of DNA probes, the sample will bind to thoseprobes that are complementary to a target DNA sequence. By evaluating towhich probes the sample DNA hybridizes more strongly, it can bedetermined whether a known sequence of DNA is present or not in thesample DNA.

The DNA microarray may be fabricated by a high-speed robotic system,generally on glass but sometimes on nylon substrates. A large number ofDNA probes may be produced on a chip using photolithographic fabricationtechniques and solid-phase chemical synthesis.

SUMMARY

A DNA probe synthesis system may include a target holder for holding atarget chip, an ejector array chip, a wash and dry station, andconveyance means for moving the target holder (and target chip) betweenthe ejector array chip and the wash and dry station. The target chip mayinclude a number of DNA probe sites, each chemically activated toreceive a sequence of DNA bases from ejectors in the ejector array.

The ejector array may be formed on a substrate, e.g., silicon, usingMicro-Electro-Mechanical Systems (MEMS) fabrication techniques. Theejector array chip may include an array of ejectors, reservoirs forcontaining the four DNA bases, and channels extending between thereservoirs and ejectors. The ejectors may be Self Focusing AcousticTransducers (SFATs). The channels may cross each other at cross points.The channels may include channels on top of the substrate with wallsformed from a photoresist material and channels recessed in thesubstrate. Alternatively, all of the channels may be recessed in thesubstrate. At a cross point, one channel may include a bridge-likeportion and the other channel may include a cavity below the bridge-likeportion.

A DNA microarray, or genome chip, may be produced using the DNA probesynthesis system. The target chip may be aligned over the ejector array.Ejectors may eject droplets of DNA bases onto DNA probe sites on thetarget chip. The target chip may then be transferred to a wash and drystation where uncoupled bases may be washed off and the target chipdried. This cycle may be repeated until desired DNA sequences (DNAprobes) are produced at the DNA probe sites.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a DNA probe synthesis system.

FIG. 2 is a flowchart describing a DNA probe synthesis operation.

FIG. 3 is a perspective view of a Self Focusing Acoustic Transducer(SFAT) with an ejection trajectory perpendicular to a liquid surface.

FIG. 4 is a perspective view of an SFAT with an ejection trajectory atan oblique angle to the liquid surface.

FIG. 5 is a perspective view of another DNA probe synthesis system.

FIG. 6 is a perspective view of an SFAT array.

FIG. 7 is a perspective view of a micro-channel structure.

FIG. 8 shows fabrication stages for producing a micro-channel structure.

FIG. 9 is a perspective view of another micro-channel structure.

FIGS. 10A-10D show fabrication stages for producing the micro-channelstructure of FIG. 9.

FIG. 11 is a perspective view of an SFAT array chip and a controlcircuit board.

FIG. 12 is a perspective view of an SFAT.

FIG. 13 is a perspective view of a switch for an SFAT.

FIG. 14 is a perspective view of another switch for an SFAT.

FIG. 15 is a perspective view of yet another switch for an SFAT.

DETAILED DESCRIPTION

FIG. 1 shows a DNA probe synthesis system 100 which may be used tosynthesize DNA probes on a target chip 105, e.g., a glass, plastic, orsilicon chip. The target chip may include an array of circular spotswhich may serve as DNA probe sites. The system 100 may include anejector array chip 110. The ejector array chip may be a silicon chipwith an array of ejectors along with microchannels and four reservoirsfor the four DNA bases, adenine (A), cytosine (C), guanine (G), andthymine (T). The ejectors may be used to eject microdroplets of the fourDNA bases in any desired sequence. The system may also contain a washand dry station 115, servomotors to slide the target glass between theejector array and the wash and dry station, and control electronics. Thesystem 100 may be linked to a computer for data input and to water andnitrogen sources for washing and drying.

FIG. 2 is a flowchart describing a DNA probe synthesis operation 200.The DNA probe sites on the target chip 105 may be activated for chemicalcoupling with any of the four DNA bases (block 205). An exemplary targetchip 105 may include 50-100 circular spots of about 1 μm diameter whichserve as the DNA probe sites. The target chip 105 may be aligned to theejector array chip 110 (block 210). DNA base droplets of about 2-5 μm indiameter may be ejected onto the target chip 105 until all the 50-100spots on the target chip 105 are covered with the desired DNA bases(block 215). Each spot may be handled by four ejectors for the four DNAbases so that any of the four bases can be ejected to the 50-100 spots.The target chip 105 may then be moved to the wash/dry section (block220). The uncoupled bases (i.e., the bases that are not coupled to theactivated spots on the target chip 105) may be washed away (block 225)and the chip dried (block 230). The target chip 105 may then be movedback to and aligned again with the ejector array chip 110, which ejectsanother set of desired DNA bases over the 50-100 spots, followed byanother washing/drying operation to remove the uncoupled bases. Thisprocess may be repeated until the desired DNA sequences of the DNAprobes are obtained over the 50-100 spots. If each cycle takes less than5 seconds, the total time needed to produce DNA probes including 10,000base oligonucleotides may be less than 14 hours.

The ejectors may be Self Focusing Acoustic Transducers (SFATs), such asthe SFAT 300 shown in FIG. 3. The SFAT 300 may be a heatless,nozzleless, and heatless ejector which focuses acoustic waves throughconstructive wave interference. The SFAT may be built on abulk-micromachined diaphragm with a piezoelectric ZnO film 305. The SFATmay include a set of complete annular rings 310, which act ashalf-wave-band sources to produce an intensified acoustic radiationpressure (at the focal point) which may be directed perpendicularly tothe liquid surface. The annular rings 310 may be made of aluminum andact as an electrode. The acoustic radiation pressure at the liquid-airinterface may be raised high enough to eject liquid droplets from theliquid (contained in the micromachined cavity adjacent to thediaphragm). An SFAT has been observed to eject DI water droplets lessthan 5 μm in diameter with RF pulses of 10 μsec pulsewidth.

When the annular rings are sectored as shown in FIG. 4, the acousticradiation pressure at the focal point is unbalanced in the plane of theliquid top surface, and the droplet is ejected in a direction 405 thatis oblique to the liquid surface plane. As the annular rings are carvedout with a pie shaped opening 410, the vertical displacement becomesless intensified, while the lateral displacement becomes larger at thefocal point, causing ejection to occur at an oblique angle. Thetrajectory of ejection of SFATs in the array may be controlled byselecting the size and orientation of the opening 410.

With the ejector array chip 110 shown in FIG. 1, a spot on the targetchip 105 may be covered by four SFATs located close to each other withdirectional ejections or by moving the target chip 105. For example,with four SFATs per spot, a 14×14 SFAT array may be used to cover 49spots, and a 20×20 SFAT array may be used to cover 100 spots.

In an alternative embodiment shown in FIG. 5, four sets of SPAT arrays501-504 may be provided for ejecting the four DNA bases using the fourejectors 511, 512, 513, 514, respectively. The target chip 105 may bemoved over the four SPAT arrays in sequence to deposit any of the fourbases on any spot on the target chip 105 before a washing/dryingoperation.

FIG. 6 shows a silicon chip 600 that contains a 6×6 ring SFAT array (for3×3 DNA probes) and a microfluidic transportation system for the fourDNA bases. The compact design includes four reservoirs 610-613 for thebases. Each array block may include 2×2 ring SFATs 615, each of whichcontains different DNA bases. In delivering the DNA bases from thereservoirs to each of the designated SFATs, there may be manyintersections among the microfluidic channels 620.

As shown in FIG. 7, the channels may include channels 705 formed on thesilicon surface with a photoresist material such as SU-8, a negative,epoxy-type, near-UV photoresist manufactured by the MicroChem Corp. ofNewton, Mass. The channels may also include bulk/surface micromachinedchannels 710 underneath the surface of the chip. A protruding film withseveral hundred μm² circular hole in the center may be provided over thecavity of each SFAT. The protruding film may provide the necessaryforce, through surface tension, to draw liquid from a reservoir throughthe channel into the SFAT. The protruding film may also keep the liquidlevel at the focal plane of the transducer. Each reservoir for the fourDNA bases may have a protruding film near its outlet channel to maintainthe liquid near the channel as the liquid is consumed.

FIG. 8 shows stages of a fabrication process to form an SFAT array withbuilt-in microchannels and reservoirs. A (100) silicon wafer may beoxidized. The oxide film 805 may be patterned to define chamber andchannel areas and then etched 5 μm deep in Potassium Hydroxide (KOH).Polysilicon 810 may be deposited and patterned on the wafer, followed byan LPCVD (Low Pressure Chemical Vapor Deposition) low-stresssilicon-nitride 815 (0.8 μm thick) deposition and patterning. The SFATsmay be formed by evaporating and patterning a 0.5 μm thick Al layer onthe bottom side of the wafer, which includes unpatterned siliconnitride. A 3-10 μm thick ZnO film may be deposited and 0.5 μm thick Alevaporated and delineated on the bottom of the wafer. A thickphotoresist 820, such as SU-8, may be spin-coated and patterned to form70 μm high vertical side walls for the microfluidic channel on the wafersurface opposite the SFATs. The wafer may be etched in KOH to releasethe diaphragms for the SFATs and to form under-channels 825. The waferside containing the layers for the SFATs may be protected by amechanical jig during this KOH etching.

Different etch depths for the channels and chambers may be obtained withone mask and one etching step by exploiting the markedly differentlyetch rates for (111) and (100) planes. One of the two (111) planesforming the convex corner may be covered with an etch mask (e.g.,silicon nitride for KOH). By protecting one of the (111) planes at theconvex corner, very clean and sharp corners may be obtained.

An etch mask material for KOH, 0.03 μm thick silicon nitride may bedeposited as an etch mask material, and patterned on both (100) and(111) planes to define the crossing channel area. To pattern the siliconnitride over a 100 μm deep V-groove, photoresist may be sprayed over thenitride through a nozzle for conformal coating of photoresist, becausespin coating of photoresist over a deep etch trench may produce anuneven thickness. Spin-coated photoresist is usually much thinner oversharp corners than over a flat surface, mainly due to the surfacetension. Moreover, the photoresist tends to reflow down along the (111)sidewall during its softbake. Thus, spin coating of photoresist over adeep trench may produce extremely thin photoresist near the convexboundaries of the trench, while excessive amounts of photoresistaccumulate on the bottom of the trench. To overcome this problem, aspray coating technique may be used, and photoresist may be sprayedupward to a face-down wafer. The wafer may be moderately heated toreduce the viscosity of the applied photoresist. A softbake may be donefor 10 minutes at 100° C. with the wafer in an upside-down position tokeep the thickness at the top convex corner constant during thesoftbake.

The clean convex corners at the cross of two deep channels may beobtained after two steps of long anisotropic etching in KOH at 80° C. A100 μm deep channel that runs from the northwest to the southeast isfirst formed, followed by deposition and patterning of silicon nitrideetch mask. A subsequent second KOH etching produces the crossing channelthat runs from the northeast to the southwest. The size of the openingarea at the cross (between the two channels) depends onphotolithographically-defined pattern of the silicon nitride on thesidewall. Thus, the height of the “channel gate” can be well defined,independent of the etching times. A triangular pattern of siliconnitride may be used on the (111) surface to define the crossing channelarea. Independent of the mask shape, freshly-exposed (111) planes meetthe protected (111) plane after a long enough etching. These (111)surfaces form convex corners under the silicon nitride protection layer,and act as an etch stop. Consequently, protected convex corners are verystable in a silicon anisotropic wet etchant.

In an alternative ejector array structure, a two-story separationstructure may be provided at the cross point of two different channelscarrying different liquids to separate the liquids. As illustrated inFIG. 9, one liquid may pass along an overhanging bridge-like channel900, while the other liquid may pass through under the bridge-likechannel 905.

FIGS. 10A-10D show stages of a fabrication process to form an SFAT arraywith a two-story structure. A 0.03 μm thick silicon nitride may bedeposited and patterned on a (100) silicon wafer, which is then etchedin KOH at 80° C. to produce 100 μm deep V-grooves 1005 for the columnchannels. Another 0.03 μm thick silicon nitride may be deposited overthe wafer surface, including the etched surface, to protect potentialconvex corners. This silicon nitride layer may be patterned to definethe row channels 1010. After a second KOH etching to produce the rowchannels, silicon nitride may deposited on the wafer surface to protectthe formed channels.

The silicon nitride may be patterned for the small chamber areas (underthe overhanging bridge-like channels) at the channel crosses 1015,followed by conformal deposition and patterning of 2 μm thicksacrificial polysilicon. A 2 μm thick low-stress LPCVD silicon-nitridemay be deposited over the wafer surface and patterned. The wafer may beetched in KOH to release the silicon-nitride overhanging channel 1020that crosses over an etched cavity, which may act an under-channel 1025.The cavity may be 100 μm deep from the bottom of the overhangingchannel. The low-stress LPCVD silicon-nitride layer may be used as anoverhanging bridge and also as an isolation layer for two differentliquids.

A flip-chip bonding technique using indium bumps may be used to attachthe SFAT array chip 1105 to a control circuit board 1110, as shown inFIG. 11. The flip-chip approach is scalable in that the array size canbe increased indefinitely without any difficulty on I/O to and from thecontrol circuit. The length of the connecting wire between the ejectorarray and the control circuit may also be reduced.

Since the ring SFATs may operate at 300-600 MHz, electromagneticinterference (EMI) among the SFATs may need to be shielded out. As shownin FIG. 12, coaxial-shape waveguide indium bumps 1205 and conductivewalls 1210 may be used to shield each SFAT from the EMI. The conductivewalls may act as a common ground. Indium may also be used for the wallto save extra fabrication steps.

The control board may generate high voltage (around 90 V peak-to-peak),pulsed sinusoidal signals to drive the SFATs individually and withoutcross talk between the SFATs. Alternatively, MEMS switches 1300-1302,such as those shown in FIGS. 13-15, may be provided at each SFAT to turna continuous sinusoidal wave on and off. The MEMS switches may include aground line 1305, an RF input 1310, and RF output 1315, and a movablemetal plate 1320. A 300-600 MHz continuous sinusoidal wave may beapplied to the entire SFAT chip. The MEMS switches may be turned on andoff to deliver pulsed sinusoidal waves to each SFAT. The MEMS switchesmay be integrated with SFATs on a single chip through surfacemicromachining. These MEMS switches may simplify the control electronicsfor a two-dimensional SFAT array.

The DNA synthesis system 100 may be used to fabricate a DNA microarray,or genome chip, on demand. The DNA microarray may be used tocharacterize single nucleotide polymorphisms (SNPs). For example, a DNAmicroarray may be used to characters SNPs in 5-10 candidate genes eachrelevant to common public health problems such as heart disease, colon,breast and prostate cancer. Each gene may contain multiple SNPs, fivebeing a good estimate. Therefore, 25-50 variants can be analyzed, eachrequiring two alleles for a total of 50-100 probes.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the invention. For example, blocks in theflowchart may be skipped and/or performed out of sequence and stillproduce desirable results. Accordingly, other embodiments are within thescope of the following claims.

1. Apparatus comprising: a substrate including a plurality of targetlocations, a plurality of reservoirs, each reservoir containing one of aplurality of DNA base materials and including at least four reservoirs,each of said four reservoirs having a different DNA base materialtherein, a plurality respective groups of ejectors for each respectivetarget location, each respective group plurality of ejectors operatingto eject one of said plurality of DNA base materials when electricallyactuated toward one of said respective target locations, each saidejector comprising a Self Focusing Acoustic Transducer (SFAT) whichfocuses waves using constructive interference, and a plurality ofchannels, wherein each channel connects one of the respective groups ofejectors to a respective corresponding reservoir, and each said channelreceives one of said base materials from said corresponding reservoirwhen said respective group of ejectors is actuated, thus coupling saidone of said base materials from said respective reservoir to saidrespective group of ejectors.
 2. The apparatus of claim 1, wherein thesubstrate comprises a material selected from silicon, plastic, andglass.
 3. The apparatus of claim 1, wherein the ejectors are arranged ingroups of four ejectors for each target location.
 4. The apparatus ofclaim 3, wherein each of said four ejectors is operative to eject adroplet of the DNA base material to a same spot on a target chip.
 5. Theapparatus of claim 1, wherein the ejectors are arranged in four separatesub-arrays, the ejectors in each sub-array operative to eject the sameDNA base material.
 6. The apparatus of claim 1, wherein the plurality ofchannels include a first channel and a second channel including at leasta portion extending over or under the first channel without any mixingof the two liquids flowing through said channels.
 7. The apparatus ofclaim 6, wherein the first channel and the second channel extend fromdifferent ones of the plurality of reservoirs.
 8. The apparatus of claim6, wherein the first channel includes walls on a surface of thesubstrate and the second channel is recessed into the surface of thesubstrate.
 9. The apparatus of claim 6, wherein the first channel andthe second channel are recessed into a surface of the substrate, andwherein the second channel includes a chamber under the first channel.10. The apparatus of claim 1, wherein each of the plurality of ejectorsis operative to eject a droplet of the base material having a diameterof about 3-70 μm.
 11. An apparatus as in claim 1, wherein said ejectorsare formed of a plurality of conductive annular rings.
 12. An apparatusas in claim 11, wherein said annular rings are annular with a pieshapedcut out portion therein.
 13. A system comprising: a target holderholding a target chip with a plurality of DNA probe sites, each probesite including a DNA base; an ejector array comprising a substrateincluding, a plurality of ejectors, each ejector formed of a selffocusing acoustic transducer which focuses waves using constructiveinterference, said plurality of ejectors arranged in plural groups ofejectors, each group of ejectors having plural ejectors therein and eachgroup of plural ejectors operating responsive to an actuation, as agroup, to eject one of a plurality of DNA base materials onto one of theplurality of DNA probe sites, at least four reservoirs, each reservoirholding one of the plurality of base materials and each of the fourreservoirs holding a different DNA base material, and a plurality ofchannels, each channel extending between one group of the ejectors andone of the reservoirs and fluidically coupling between said one group ofejectors and said one reservoir; a wash and dry station including awashing part that washes the plurality of DNA probe sites and a dryingpart that operates to dry the plurality of DNA probe sites; and aconveyance part that conveys the target holder between the ejector arrayand the wash and dry station.
 14. The system of claim 13, wherein thesubstrate comprises a material selected from silicon, plastic, andglass.
 15. The system of claim 13, wherein each of said four ejectors isoperative to eject a droplet of the DNA base material to a same spot ona target chip.
 16. The system of claim 13, wherein the ejectors arearranged in four separate sub-arrays, the ejectors in each sub-arrayoperative to eject the same DNA base material.
 17. The system of claim13, wherein the plurality of channels include a first channel and asecond channel including at least a portion extending below or above thefirst channel.
 18. The system of claim 17, wherein the first channel andthe second channel extend from different ones of the plurality ofreservoirs.
 19. The system of claim 17, wherein the first channelincludes walls on a surface of the substrate and the second channel isrecessed into the surface of the substrate.
 20. The system of claim 17,wherein the first channel and the second channel are recessed into asurface of the substrate, and wherein the second channel includes achamber under the first channel.
 21. The system of claim 13, whereineach of the plurality of ejectors is operative to eject a droplet of thebase material having a diameter of 3-70 μm.
 22. A system as in claim 13,wherein said ejectors are formed of a plurality of conductive annularrings.
 23. A system as in claim 22, wherein said annular rings have apie shaped cutout portion therein.
 24. Apparatus comprising: a substrateincluding a plurality of reservoirs, each reservoir containing one of aplurality of DNA base materials, a plurality of ejectors, each ejectoroperating to eject one of said plurality of DNA base materials whenelectrically actuated, each said ejector comprising a Self FocusingAcoustic Transducer (SFAT) which focuses waves using constructiveinterference, and a plurality of channels, each channel connecting oneof the ejectors to one of the reservoirs, and receiving one of said basematerials from one of said reservoirs when an ejector is actuated, thuscoupling said one of said base materials from said one of saidreservoirs to an ejector, wherein each self focusing acoustic transduceris formed of a plurality of annular rings made of a conductive material.25. The apparatus of claim 24, wherein the annular rings are arranged toform an acoustic radiation pressure at a focal point which is unbalancedrelative to a plane of a top surface of the DNA material, causing theejection of DNA material to occur at an oblique angle.
 26. A systemcomprising: a target holder holding a target chip with a plurality ofDNA probe sites, each probe site including a DNA base; an ejector arraycomprising a substrate including a plurality of ejectors, each ejectorformed of a self focusing acoustic transducer which focuses waves usingconstructive interference, each ejector operating responsive to anactuation to eject one of a plurality of DNA base materials onto one ofthe plurality of DNA probe sites, a plurality of reservoirs, eachreservoir holding one of the plurality of base materials, and aplurality of channels, each channel extending between one of theejectors and one of the reservoirs and fluidically coupling between saidone ejector and said one reservoir; a wash and dry station including awashing part that washes the plurality of DNA probe sites and a dryingpart that operates to dry the plurality of DNA probe sites; and aconveyance part that conveys the target holder between the ejector arrayand the wash and dry station, wherein each self focusing acoustictransducer is formed of a plurality of annular rings made of aconductive material.
 27. The system of claim 26, wherein the annularrings are arranged to form an acoustic radiation pressure at a focalpoint which is unbalanced relative to a plane of a top surface of theDNA material, causing the ejection of DNA material to occur at anoblique angle.